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- ItemA three-dimensional ex vivo tri-culture model mimics cell-cell interactions between acute myeloid leukemia and the vascular niche(Pavia : Ferrata Storti Foundation, 2017) Bray, Laura J.; Binner, Marcus; Körner, Yvonne; von Bonin, Malte; Bornhäuser, Martin; Werner, CarstenEx vivo studies of human disease, such as acute myeloid leukemia, are generally limited to the analysis of two-dimensional cultures which often misinterpret the effectiveness of chemotherapeutics and other treatments. Here we show that matrix metalloproteinase-sensitive hydrogels prepared from poly(ethylene glycol) and heparin functionalized with adhesion ligands and pro-angiogenic factors can be instrumental to produce robust three-dimensional culture models, allowing for the analysis of acute myeloid leukemia development and response to treatment. We evaluated the growth of four leukemia cell lines, KG1a, MOLM13, MV4-11 and OCI-AML3, as well as samples from patients with acute myeloid leukemia. Furthermore, endothelial cells and mesenchymal stromal cells were co-seeded to mimic the vascular niche for acute myeloid leukemia cells. Greater drug resistance to daunorubicin and cytarabine was demonstrated in three-dimensional cultures and in vascular co-cultures when compared with two-dimensional suspension cultures, opening the way for drug combination studies. Application of the C-X-C chemokine receptor type 4 (CXCR4) inhibitor, AMD3100, induced mobilization of the acute myeloid leukemia cells from the vascular networks. These findings indicate that the three-dimensional tri-culture model provides a specialized platform for the investigation of cell-cell interactions, addressing a key challenge of current testing models. This ex vivo system allows for personalized analysis of the responses of patients’ cells, providing new insights into the development of acute myeloid leukemia and therapies for this disease.
- ItemLimbal stromal cells derived from porcine tissue demonstrate mesenchymal characteristics in vitro(London : Nature Publishing Group, 2017) Fernández-Pérez, Julia; Binner, Marcus; Werner, Carsten; Bray, Laura J.Limbal stromal cells (LSCs) from the human ocular surface display mesenchymal stromal cell characteristics in vitro. In this study, we isolated cells from the porcine limbal stroma (pLSCs), characterised them, and evaluated their ability to support angiogenesis and the culture of porcine limbal epithelial stem cells (pLESCs). The isolated cells adhered to plastic and grew in monolayers in vitro using serum-supplemented or serum-free medium. The pLSCs demonstrated expression of CD29, and cross-reactivity with anti-human CD45, CD90, CD105, CD146, and HLA-ABC. However, expression of CD105, CD146 and HLA-ABC reduced when cultured in serum-free medium. PLSCs did not undergo adipogenic or osteogenic differentiation, but differentiated towards the chondrogenic lineage. Isolated cells were also co-cultured with human umbilical vein endothelial cells (HUVECs) in star-shaped Poly(ethylene glycol) (starPEG)-heparin hydrogels to assess their pericyte capacity which supported angiogenesis networks of HUVECs. PLSCs supported the three dimensional HUVEC network for 7 days. The isolated cells were further growth-arrested and evaluated as feeder cells for pLESC expansion on silk fibroin membranes, as a potential carrier material for transplantation. PLSCs supported the growth of pLESCs comparably to murine 3T3 cells. In conclusion, although pLSCs were not completely comparable to their human counterpart, they display several mesenchymal-like characteristics in vitro.